Nomoto Mika, Tada Yasuomi
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan.
Center for Gene Research, Nagoya University, Nagoya, Aichi, Japan.
Methods Mol Biol. 2018;1830:337-349. doi: 10.1007/978-1-4939-8657-6_20.
Biotic and abiotic stimuli induce profound transcriptional reprograming in plants through sophisticated regulation of transcription factors (TFs). Recombinant proteins of TFs play an important role in unveiling their molecular functions. Cell-free protein synthesis (CFPS) system from wheat germ has been developed as one of the most efficient protein synthesis platforms. However, preparation of linear DNA templates for in vitro transcription is time-consuming and laborious. Here, we describe a versatile method for in vitro transcription and translation of the wheat germ CFPS system. Our two-step PCR method enables researchers to generate a variety of transcription templates from a single plasmid including fusion proteins of an N- or C-terminal tag and truncated proteins. Thus, this method supports a rapid and high-throughput expression of proteins for a large-scale proteomics analysis.
生物和非生物刺激通过对转录因子（TFs）的精细调控，在植物中诱导深刻的转录重编程。转录因子的重组蛋白在揭示其分子功能方面发挥着重要作用。小麦胚无细胞蛋白质合成（CFPS）系统已被开发为最有效的蛋白质合成平台之一。然而，用于体外转录的线性DNA模板的制备既耗时又费力。在这里，我们描述了一种用于小麦胚CFPS系统体外转录和翻译的通用方法。我们的两步PCR方法使研究人员能够从单个质粒生成各种转录模板，包括N端或C端标签的融合蛋白和截短蛋白。因此，该方法支持蛋白质的快速高通量表达，用于大规模蛋白质组学分析。
